Field of the Invention
The present invention relates to macroporous particles especially useful in multiplex assays and to manufacturing methods and methods of use of such particles.
Background of the Invention
Research and development of macroporous poly (styrene-divinylbenzene) microspheres has been ongoing since the late 1950's. These macroporous microspheres have the ability to interact with atoms, ions, and molecules. Generally speaking, macroporous microspheres are microspheres with pore diameter sizes exceeding about 20 nm. The fact that these macroporous microspheres are porous yields the unique ability to form interactions on the interior “pores” as well as the exterior surface of the macroporous microspheres.
Many applications of macroporous microspheres focused on use as ion-exchange resins and in liquid chromatography. Functionalization of these macroporous microspheres has led to their use as separation media. The internal makeup of the styrene/divinylbenzene co-polymer is ideal for the introduction of and reaction with compounds and molecules with known high exchange capacities. Use for macroporous microspheres has also been found in biological applications by physical adsorption of materials of interest, commonly using reverse-phase ion exchange chromatography, although other methods are possible and have been documented. Cation-exchange resins have been prepared by functionalization of macroporous microspheres with sulfonic acid, which can be useful for acid catalysis and has been well documented in the literature.
In one method for producing macroporous microspheres, synthesis is accomplished by suspension polymerization employing an inert diluent. Once polymerization is completed, the inert diluent is removed and porous structure remained within the polymer particles. In this method, the particles produced are fairly large, but polydisperse. These characteristics are not desirable for use in chromatography where flow conditions and packing efficiency are important factors.
Another method for producing uniform polymer macroporous microspheres uses seeded emulsion polymerization to produce uniform macroporous microspheres. A modification of this method uses linear polystyrene with a solvent or non-solvent type diluent and produces macroporous microspheres with some ability to control pore volume and specific surface area, depending on whether the linear polymer is combined with a solvent or non-solvent. However, relatively high levels of crosslinker are required to generate particles with high surface area.
Analysis of clinical specimens is important in science and medicine. A wide variety of assays to determine qualitative and/or quantitative characteristics of a specimen are known in the art. Detection of multiple analytes, or separately identifiable characteristics of one or more analytes, through single-step assay processes are known, but have some limitations such as the extended times typically required to enable the detection and classification of multiple analytes and the low sensitivities (low signal) achievable in assays.
A capability to perform simultaneous, multiple interrogations of a specimen in a single assay process is known as “multiplexing” and a process to implement such a capability is a “multiplexed assay.” One well known prior art technique having a multiplexed assay capability is flow cytometry. Flow cytometry is an optical technique that analyzes particular particles in a fluid mixture based on the particles' optical characteristics using an instrument known as a flow cytometer. Background information on flow cytometry may be found in Shapiro, “Practical Flow Cytometry,” Third Ed. (Alan R. Liss, Inc. 1995); and Melamed et al., “Flow Cytometry and Sorting,” Second Ed. (Wiley-Liss 1990), which are incorporated herein by reference.
Flow cytometers hydrodynamically focus a fluid suspension of particles into a thin stream so that the particles flow down the stream in substantially single file and pass through an examination zone. A focused light beam, such as a laser beam illuminates the particles as they flow through the examination zone. Optical detectors within the flow cytometer measure certain characteristics of the light as it interacts with the particles. Commonly used flow cytometers such as the assay system commercially available from Luminex Corporation of Austin, Tex. can measure forward light scatter (generally correlated with the refractive index and size of the particle being illuminated), side light scatter (generally correlated with the particle's size), and particle fluorescence at one or more wavelengths.
Another example of such a multiplex assay system is the xMAP® technology that is commercially available from Luminex Corporation of Austin, Tex. The xMAP technology uses a family of dyed particles onto which one or more assay-specific reagents may be applied (e.g., by coupling to one or more functional groups on the surface of the particles). The particle platform employs different sets of particles distinguishable by fluorescence. For example, the sets of particles may be distinguishable by wavelength of fluorescence, intensity of fluorescence, ratio of intensities of fluorescence at different wavelengths, etc. In general, the variation of fluorescence may be integrated by incorporating different dyes and/or fluorophores into the particles and/or coupled to a surface of the particles. In some embodiments, the sets of particles may be additionally distinguishable by size and/or shape. In any case, a particle platform having distinguishable carrier particles is generally advantageous because it uses fluid based kinetics to bind several different analytes to the assay-specific reagents.
In general, each of the different sets of particles may have a different reagent coupled thereto. The different reagents may selectively react with different analytes in a fluid sample. In other words, each of the different reagents may react with one analyte in a sample, but may not substantially react with any other analytes in the sample. In some cases, one or more additional detectable reagents may be allowed to react with one or more of the analytes. The one or more additional reagents may be detectable (and possibly distinguishable) by fluorescence (e.g., wavelength of fluorescence, intensity of fluorescence, etc.). In addition to the enhanced reaction kinetics, the use of a multiplexed assay platform advantageously allows a user to simply add or remove one or more subsets of particles, to or from the population to which the sample is exposed, to vary the analytes being investigated.
The above mentioned techniques and methods fail to take advantage of a macroporous microsphere in a quantitative single or multiplexed biological assay system.
Tuncel et al., “Electron microscopic observation of uniform macroporous particles. I. Effect of seed latex type and diluent,” Journal of Applied Polymer Science, Vol. 71, No. 14, 1999, pp. 2271-2290; U.S. Pat. No. 4,459,378 to Ugelstad; and El-Aasser et al., “Synthesis and Characterization of Monodisperse Porous Polymer Particles,” Journal of Polymer Science: Part A, Vol 30, 1992, pp. 235-244, describe methods of synthesizing macroporous microspheres. U.S. Pat. Nos. 7,141,431; 5,981,180; 6,632,526; 6,733,812; 7,241,883; and 7,274,316 describe various systems and methods for multiplexed biological assays. U.S. Pat. No. 6,773,812 describes methods of manufacture ad use of magnetically-responsive beads. All references cited herein are incorporated by reference as if fully set out herein to the fullest extent permissible by law or regulation.